presynapticdiversityrevealedbyca2 -permeableampa … · the well characterized, cp-ampar-selective...

Cellular/Molecular

Presynaptic Diversity Revealed by Ca2�-Permeable AMPAReceptors at the Calyx of Held Synapse

Brendan Lujan, Andre Dagostin, and X Henrique von GersdorffVollum Institute, Oregon Health and Science University, Portland, Oregon 97239

GluA2-lacking Ca 2�-permeable AMPARs (CP-AMPARs) play integral roles in synaptic plasticity and can mediate excitotoxic cellularsignaling at glutamatergic synapses. However, the developmental profile of functional CP-AMPARs at the auditory brainstem remainspoorly understood. Through a combination of electrophysiological and live-cell Ca 2� imaging from mice of either sex, we show that thesynaptic release of glutamate from the calyx of Held nerve terminal activates CP-AMPARs in the principal cells of the medial nucleus of thetrapezoid body in the brainstem. This leads to significant Ca 2� influx through these receptors before the onset of hearing at postnatal day12 (P12). Using a selective open channel blocker of CP-AMPARs, IEM-1460, we estimate that �80% of the AMPAR population arepermeable to Ca 2� at immature P4 –P5 synapses. However, after the onset of hearing, Ca 2� influx through these receptors was greatlyreduced. We estimate that CP-AMPARs comprise approximately 40% and 33% of the AMPAR population at P18 –P22 and P30 –P34,respectively. By quantifying the rate of EPSC block by IEM-1460, we found an increased heterogeneity in glutamate release probability foradult-like calyces (P30 –P34). Using tetraethylammonium (TEA), a presynaptic potassium channel blocker, we show that the apparentreduction of CP-AMPARs in more mature synapses is not a consequence of presynaptic action potential (AP) speeding. Finally, throughpostsynaptic AP recordings, we show that inhibition of CP-AMPARs reduces spike fidelity in juvenile synapses, but not in more maturesynapses. We conclude that the expression of functional CP-AMPARs declines over early postnatal development in the calyx of Heldsynapse.

Key words: AMPA; auditory brainstem; laser confocal Ca imaging; calyx of Held; EPSC; MNTB

IntroductionAMPARs mediate the majority of fast excitatory transmission inthe CNS. They are vital to CNS development and proper function(Traynelis et al., 2010). AMPARs are tetrameric ligand gated ion

channels composed of GluA1– 4 subunits that open in responseto glutamate (Dingledine et al., 1999). GluA2-lacking AMPARsrender the channel complex permeable to Ca 2� and are thusreferred to as Ca 2�-permeable AMPARs (CP-AMPARs; Holl-mann et al., 1991; Kuner et al., 2001). GluA2-containingAMPARs are impermeable to Ca 2� because of the single aminoacid Q/R posttranscriptional editing site in the mRNA that en-codes the channel pore (Burnashev et al., 1992). The majority ofGluA2 subunits are edited in the brains of rodents and humans(Hume et al., 1991; Carlson et al., 2000) and animals deficient inthe Q/R site editing die prematurely (Brusa et al., 1995). In severalbrain regions, CP-AMPARs serve important functions in the in-duction and modulation of activity-dependent synaptic plasticityat developing and mature synapses (Jia et al., 1996; Plant et al.,

Received Oct. 4, 2018; revised Dec. 14, 2018; accepted Jan. 3, 2019.Author contributions: B.L. wrote the first draft of the paper; B.L., A.D., and H.v.G. edited the paper; B.L., A.D., and

H.v.G. designed research; B.L. and A.D. performed research; B.L., A.D., and H.v.G. analyzed data; B.L., A.D., and H.v.G.wrote the paper.

This work was supported by the National Institute on Deafness and Other Communication Disorders–NationalInstitutes of Health (Grants DC012938 and F32-DC017644). We thank Drs. Victor Derkach and Dale Fortin for discus-sions and for first suggesting the use of IEM-1460.

The authors declare no competing financial interests.Correspondence should be addressed to Henrique von Gersdorff at [email protected]://doi.org/10.1523/JNEUROSCI.2565-18.2019

Copyright © 2019 the authors

Significance Statement

The calyx of Held synapse is pivotal to the circuitry that computes sound localization. Postsynaptic Ca2� influx via AMPARs maybe critical for signaling the maturation of this brainstem synapse. The GluA4 subunit may dominate the AMPAR complex atmature synapses because of its fast gating kinetics and large unitary conductance. The expectation is that AMPARs dominated byGluA4 subunits should be highly Ca2� permeable. However, we find that Ca2�-permeable AMPAR expression declines duringpostnatal development. Using the rate of EPSC block by IEM-1460, an open channel blocker of Ca2�-permeable AMPARs, wepropose a novel method to determine glutamate release probability and uncover an increased heterogeneity in release probabilityfor more mature calyces of Held nerve terminals.

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2006; Kim and von Gersdorff, 2016; Park et al., 2016). Addition-ally, Ca 2� permeability through this receptor regulates neuronalCa 2�-induced excitotoxicity (Carriedo et al., 1998; Noh et al.,2005; Sebe et al., 2017) and the progression of several CNS pa-thologies are dependent on Ca 2� influx through these receptors(Weiss, 2011).

Mature auditory brainstem synapses possess the unique abil-ity to maintain extremely fast and temporally precise informationtransmission, processes thought to be crucial for sound localiza-tion (Spirou et al., 1990; Kopp-Scheinpflug et al., 2011). Imma-ture synapses are not immediately able to meet these demandsand several synaptic specializations occur over the course of post-natal development that promote fast and indefatigable transmis-sion (Taschenberger et al., 2002; Krachan et al., 2017). The calyxof Held nerve terminal synapses onto the principal cells in themedial nucleus of the trapezoid body (MNTB), a critical compo-nent of the ascending auditory pathway (Kandler and Friauf,1993; Portfors and von Gersdorff, 2013). The principal cells of theMNTB undergo a developmental switch in the composition ofthe postsynaptic AMPARs to include GluA4 subunits; which havefast gating kinetics, large unitary conductance and rapidly re-cover from desensitization (Joshi et al., 2004; Koike-Tani et al.,2005). A population of AMPARs dominated by the GluA4 sub-unit would be expected to possess high Ca 2� permeability, butexperiments to test the hypothesis directly have not been per-formed after functional maturity. Indirect measurements of theAMPAR subunit composition assayed by measuring the rectifi-cation index from EPSC IV curves recorded in the presence ofintracellular polyamines suggest that these receptors should behighly permeable to Ca 2� because of the strong rectification.However, direct measurements of the relative levels of GluA2subunits, the critical regulator of Ca 2� permeability, are not incomplete agreement (Caicedo and Eybalin, 1999; Joshi et al.,2004; Koike-Tani et al., 2005). Additionally, using a CP-AMPARspecific pharmacological antagonist, �50% of the EPSC ampli-tude was blocked when normalized to control in experimentsperformed in postnatal day 14 (P14) mice (Joshi et al., 2004). Thiswould be an unexpected result if CP-AMPAR expression re-mained high throughout development. Therefore, the expressionprofile of CP-AMPARs over the course of development requiresfurther investigation.

Here, we examined the functional expression of CP-AMPARsover the time course of hearing onset in the mouse MNTB. Usingthe well characterized, CP-AMPAR-selective open-channel blockerIEM-1460 (Twomey et al., 2018), we demonstrate with patch-clamp electrophysiology and live-cell Ca 2� imaging that CP-AMPAR expression declines with postnatal development. Thiswas an unexpected finding because the majority of AMPARs areGluA4 containing at mature synapses and presumed to be Ca 2�

permeable. Additionally, we performed recordings of high-frequency action potential (AP) trains and found that the inhibi-tion of CP-AMPARs did not affect the spiking fidelity in matureanimals, whereas a strong reduction was observed in juvenileanimals. We conclude that the Ca 2� permeability throughAMPARs declines with postnatal development and suggest thatmost functional AMPARs must contain at least one GluR2 sub-unit together with GluR4 subunits at mature synapses.

Materials and MethodsAnimals. All animal procedures performed in this study were approvedby the Institutional Animal Care and Use Committee of Oregon Healthand Science University. C57BL/6J mice (The Jackson Laboratory) of ei-ther sex were used at four postnatal day time points: P4 –P5, P8 –P11,

P18 –P22, and P27–P34. Veterinary care was provided by the Depart-ment of Comparative Medicine of Oregon Health and Science Universityand animals were housed in the onsite vivarium. Data were acquiredfrom a total of 83 animals.

Electrophysiology. Brainstem slices were prepared as described previ-ously (Lujan et al., 2016). Briefly, animals were anesthetized and killedbefore the brain was quickly removed and placed in an ice-cold cuttingartificial CSF (ACSF). The cutting solution contained the following (inmM): 85 NaCl, 2.5 KCl, 25 glucose, 25 NaHCO3, 1.25 NaHPO4, 75 su-crose, 0.5 CaCl2, 7 MgCl2, 3 myo-inositol, 2 Na2-pyruvate, and 0.4 ascor-bic acid; pH 7.3 when bubbled with carbogen gas (95% O2-5% CO2). Themeninges were removed under a dissection microscope and the brains-tem was mounted on a plate for slicing with a vibratome (VT1200S; LeicaMicrosystems). Transverse brainstem slices containing the MNTB weremade at a thickness of 200 �m and were transferred to an incubationchamber filled with normal ACSF bubbled with carbogen gas for 30 – 60min at 37°C. The normal ACSF contained the following (in mM): 125NaCl, 2.5 KCl, 25 glucose, 25 NaHCO3, 1.25 NaHPO4, 1.2 CaCl2, 1MgCl2, 3 myo-inositol, 2 Na2-pyruvate, and 0.4 ascorbic acid, osmolarity:310 –320 mOsm and pH 7.3 when bubbled with carbogen gas. Slices weremaintained thereafter in normal ACSF (up to 6 h) at room temperature(23–25°C).

Brainstem slices were transferred to a recording chamber and placedunder a BX51WI (Olympus) or Axioskop 2FS (Zeiss) microscope. Theslices were continuously perfused at 1–2 ml/min with normal ACSF bub-bled with carbogen gas. Experiments were performed at room tempera-ture (23–25°C) unless otherwise noted. For experiments performed atmore physiological temperature (PT; 33–35°C), the bath solution tem-perature was regulated by an inline heater and warmed platform (WarnerInstruments). Neurons were visualized by infrared-differential interfer-ence contrast microscopy with a 60�/1.0 numerical aperture water-immersion objective (Olympus) connected to a CCD camera (QI-Click;QImaging). Whole-cell voltage- or current-clamp recordings were madeusing a HEKA EPC-10/2 amplifier controlled by Patchmaster software(HEKA). Data were acquired at 100 kHz and low-pass filtered at 2.9 kHz.Patch pipettes were pulled from standard borosilicate capillary glass(WPI) using a P97 puller (Sutter Instruments). Recording pipettes had2.0 – 4.0 M� open-tip resistance in the bath solution. Electrophysiologydata were analyzed offline using custom-written routines in IGOR Pro(WaveMetrics).

Postsynaptic voltage-clamp recordings were made from the principalcells of the MNTB using an internal solution with the following compo-sition (in mM): 130 K-gluconate, 20 KCl, 5 Na2 phosphocreatine, 10HEPES, 5 EGTA, 4 Mg-ATP, 0.5 GTP, and 5 QX 314 adjusted to pH of 7.3with KOH and osmolarity of 300 –310 mOsm. To evoke EPSCs, a bipolarstimulating electrode was placed near the midline and 100 �s squarepulses were applied (1–100 Hz) through an isolated stimulator (modelISO-Flex; AMPI) controlled by a Master-8 stimulator or directly throughPatchmaster. To isolate the AMPAR-mediated EPSCs, we added 0.5 �M

strychnine to the bath solution to inhibit glycine receptors and 10 �M

bicuculline to inhibit GABAA receptors. The principal cells were held at acommand voltage of �70 mV and the liquid junction potential was leftuncorrected. The series resistance (Rs) was monitored throughout allexperiments and online compensated electronically to 1 M�. Cells withuncompensated Rs � 8 M� or leak current ��200 pA were excludedfrom analysis. No Rs compensation was used for recording of spontane-ous mEPSC events to avoid additional noise. Postsynaptic current-clamprecordings were made from the principal cells of the MNTB using thesame internal solution used for voltage-clamp recordings except thatQX-314 was excluded from the pipette. All current-clamp recordingswere performed at PT (33–35°C) and principal cells were current injectedto set the resting membrane potential to �70 mV (mean current injec-tion �75.3 29.4 pA; n 14 cells), which is close to the in vivo restingpotential of MNTB principal cells (Lorteije et al., 2009). The compoundsQX-314 and IEM-1460 were from Tocris Bioscience. OGB1 was fromInvitrogen. All other reagents were purchased from Sigma-Aldrich.

Ca2� imaging. To observe changes in [Ca 2�]i, the EGTA concentra-tion was reduced to 0.0 or 0.5 mM and the Ca 2� indicator Oregon GreenBapta-1 (OGB1; Kd �170 nM) was added to the internal pipette solution

2982 • J. Neurosci., April 17, 2019 • 39(16):2981–2994 Lujan et al. • Auditory Ca2�-Permeable AMPA Receptors

(50 �M). To ensure that the dye was maximally dialyzed into the cells,experiments were initiated 5 min after achieving whole-cell configura-tion. An initial voltage step was then applied to all cells and used as acontrol for dye loading. Synaptic activity was driven by midline stimula-tion and the Ca 2� fluorescence was recorded using an Evolve 512EMCCD camera (Photometrics) attached to a spinning disk confocal(Yokogawa model CSU-X1) on a BX51WI microscope (Olympus) con-trolled by Slidebook 6 software (Intelligent Imaging Innovations). Thesampling rate was 33 Hz with a 30 ms exposure time and the acquisitionwas TTL triggered by Patchmaster software. Fluorescence data were mea-sured as �F/F0 from selected regions of interest (cell soma whereAMPARs are located) using Slidebook 6 and plotted as a function of time.The EPSC responses were recorded simultaneously in voltage-clampholding at �70 mV. Importantly, at this holding potential, NMDA re-ceptors are nearly completely blocked and voltage-gated Ca 2� channelsare closed (Bollmann et al., 1998).

Experimental design and statistical analysis. All data are presented asmean SEM. Replicate numbers (n) are presented for all experimentalconditions and refer to the numbers of cells recorded per condition.Once a slice was used for an experiment, it was discarded and fresh sliceswere used for subsequent experiments. Statistical analysis was performedusing GraphPad Prism 7.03 software for Windows. Graphs were alsobuilt in Prism and sample traces were constructed in Igor Pro. The sta-tistical significance level was set at p � 0.05 and is denoted by asterisks inthe figures (*p � 0.05, **p � 0.005, ***p � 0.001); exact values arepresented in the figure legends. The appropriate statistical tests were usedto make comparisons between groups. A two-tailed t test was used todetermine statistical significance when comparing two groups. One-wayANOVA was used to compare three groups. If a one-way ANOVA testwas used, a post hoc multiple comparisons Tukey’s test was used to com-pare the mean of one group with the mean of every other group. Datafollowed binomial distributions and parametric tests were used in allscenarios.

ResultsIsolating Ca 2� permeable EPSCs with IEM-1460The fast EPSCs of the MNTB principal cells are mediated byAMPARs (Forsythe and Barnes-Davies, 1993; Yang et al., 2011).To determine the contribution of CP-AMPARs to postsynapticcurrents during postnatal development, we recorded EPSCs fromthe principal cells of the MNTB at four developmental age ranges.We chose age ranges that span the development of postnatal hear-ing in the mouse. During this period, the postsynaptic AMPARsubunit composition becomes specialized to ensure fast andhigh-fidelity transmission. EPSCs were evoked at a frequency of 1Hz and an activity-dependent block of the EPSC was observed inthe presence of IEM-1460, a selective open-channel antagonist ofGluA2-lacking AMPARs (Fig. 1A,B; Sebe et al., 2017; Twomey etal., 2018). IEM-1460 blocked 78.1 5.1% of the EPSC amplitudein immature P4 –P5 animals, whereas a progressively smaller ef-fect of the drug on the EPSC amplitude was observed as the age ofthe animals increased (P8 –P11: 68.0 2.6%, P18 –P22: 40.8 2.7%, P30 –P34: 33.3 5.0%; Fig. 1C,D). Importantly, the IEM-1460 block was fully reversible (Fig. 1A,B).

We also performed similar experiments in P18 –P22 animals,but used the high-affinity competitive AMPAR antagonist NBQX(1 �M). Recordings in the presence of NBQX led to a rapid andnear complete block of the EPSC (99.3 0.1%; Fig. 1A). IEM-1460 and NBQX have similar molecular weights (454 Da forIEM-1460 and 380 Da for NBQX) and should reach the synapticcleft within our brain slices at similar rates (Fig. 1A). We charac-terized the time course of the CP-AMPAR block by fitting a singleexponential function to the decay of the EPSC amplitude plottedagainst time for all age groups (Fig. 1A). In P4 –P5 animals, theaverage � was fast: 1.4 0.1 min. As the age of the animalsincreased, we observed a progressive slowing in the rate of CP-

AMPAR block (P8 –P11: 1.3 0.1 min, P18 –P22: 1.7 0.2 min,P30 –P34: 2.8 0.4 min). Like MK-801, IEM-1460 is an openchannel blocker and the rate of postsynaptic receptor block ispartly due to presynaptic glutamate release probability (Pr),whereby synapses with high Pr will possess a relatively faster �(Rosenmund et al., 1993). Therefore, we used the � of postsynap-tic CP-AMPAR block to estimate the relative presynaptic Pr (Fig.1E). In juvenile animals, the � was fastest and showed little vari-ability. However, upon maturation, the average � was slowed anddisplayed higher variability, which may reflect the intrinsic het-erogeneity of Pr for distinct calyceal morphologies in adult-likecalyces (P20 –P30; Grande and Wang, 2011). This confirms pre-vious findings of higher Pr in young synapses (P8 –P10) com-pared with more mature (P14 –P15) synapses (Taschenbergerand von Gersdorff, 2000; Iwasaki and Takahashi, 2001).

The large diversity of presynaptic Pr values that we uncoveredfor different calyx synapses at P30 –P34 may reflect differences inactive zone proteins, Ca 2� channel expression, and the su-perpriming of a subset of docked vesicles (Chen et al., 2015;Taschenberger et al., 2016). Differences in the spontaneous andsound-evoked spike activity among in vivo calyx synapses duringpostnatal development may drive these changes in Pr, as well aschanges in presynaptic calyx morphology and AMPAR expres-sion (Tokuoka and Goda, 2008; Ford et al., 2009).

These results suggest that CP-AMPAR-mediated synaptic trans-mission declines during postnatal development at the principal cellsof the MNTB. Although CP-AMPAR-mediated transmission con-tinued to decline up to P30–P34, the largest difference observed overthe developmental windows that we assayed was between the P8–P11 (prehearing) and P18–P22 (posthearing) groups. Therefore,our subsequent experiments focused on these age ranges.

To further investigate the possibility that CP-AMPAR medi-ated synaptic transmission declines with synapse maturation, weinvestigated whether spontaneous miniature EPSC (mEPSC)amplitudes were antagonized to a similar extent as the evokedEPSCs (eEPSCs) by IEM-1460 across development. Therefore,we recorded mEPSCs in control conditions and after blockingCP-AMPARS with the 1 Hz stimulation protocol in the presenceof IEM-1460 (Fig. 2A). The mEPSC amplitude was reduced by34.9 3.7% in the P18 –P22 group, similar to the effect of IEM-1460 on the eEPSC (40.8 2.7%). However, mEPSC peak am-plitude was only reduced by 42.9 2.3% in the P8 –P11 group, incontrast to the eEPSC reduction (68.0 2.6%; Fig. 2B). Weobserved no change in the frequency of events with IEM-1460 inthe P8 –P11 group or in the P18 –P22 group, suggesting no directpresynaptic effects of IEM-1460 (Fig. 2C). We then compared thepercentage block induced by IEM-1460 on the mEPSC ampli-tudes at the two developmental time points and found that it wasnot different between the two groups (Fig. 2D). To further inves-tigate the discrepancy observed between the percentage block ofIEM-1460 on the mEPSCs and eEPSCs in the P8 –P11 group, weconstructed histograms of the mEPSC peak amplitudes. The con-trol amplitude distributions (P8 –P11 and P18 –P22) were well fitwith a Gaussian function (R 2 0.9250 and R 2 0.9548, respec-tively; Fig. 2E,F). Addition of IEM-1460 to the slices produced aleftward shift to the distributions at both ages. For the IEM-1460distribution curve fits, we constrained the mean value to thatpredicted if the mEPSCs were antagonized proportionally to theeEPSCs (P8 –P11: mean was set to 21.5 pA; P18 –P22: mean wasset to 44.2 pA). Constraining the mean of the curve fits closelyapproximated our data, especially in the P18 –P22 group (R 2 0.9500). However in the P8 –P11 group, treatment with IEM-1460 shifted the distribution such that the fit invaded the

Lujan et al. • Auditory Ca2�-Permeable AMPA Receptors J. Neurosci., April 17, 2019 • 39(16):2981–2994 • 2983

distribution of electrical noise (R 2 0.6544). This result sug-gests that a population of the mEPSCs with small amplitudewere being masked by the electrical noise in the presence ofIEM-1460 in the P8 –P11 group. From these data, we concludethat the mEPSCs are probably similarly antagonized as theeEPSCs across development and that the discrepancy of per-centage block observed between mEPSCs and eEPSCs in theP8 –P11 group is likely due to an under sampling of eventswith very small amplitude. These data again suggest that post-synaptic CP-AMPAR mediated synaptic transmission declineswith postnatal development.

The frequency of control mEPSC events increased �10-foldfrom P8 –P11 to P18 –P22 (Fig. 2A,C), suggesting a larger num-ber of functional active zones at P18 –P22 synapses (Taschen-berger et al., 2002). Release probability of spontaneous vesiclefusion thus appears to increase with age, whereas the Pr of action-potential-evoked vesicle fusion decreases with age (Kavalali,2015).

High-frequency EPSC trains in IEM-1460We next investigated whether CP-AMPARs could regulate theshort-term plasticity (STP) at the developing calyx of Held syn-

apse (von Gersdorff and Borst, 2002). We recorded EPSCs elic-ited by high-frequency stimulation (HFS) trains evoked at 100 Hzin P8 –P11 and P18 –P22 animals in control conditions and afterrepetitive stimulation in the presence of IEM-1460 (Fig. 3A). Tocompare the activity-dependent effect of CP-AMPAR block onSTP, we constructed peak amplitude surface plots (Fig. 3B). TheEPSC peak amplitude is displayed as a function of stimulus num-ber with an additional z-axis representing the sweeps recordedsequentially. The control condition is portrayed as sweep 1 on thez-axis and each subsequent sweep was recorded in the presence ofIEM-1460. We found that neither the paired-pulse ratio nor the �was affected after blocking the CP-AMPARs (Fig. 3C,D). TheEPSCs were inhibited more strongly in the P8 –P11 group (57.6 8.2%) than in the P18 –P22 group (36.0 13.4%) after repetitiveHFS in the presence of IEM-1460, as evidenced in the surface plot(Fig. 3B,E,F). We compared the HFS first EPSC percentage blockto that obtained using the 1 Hz stimulation protocol in Figure 1Dto test whether the HFS train protocol equally blocked CP-AMPARs. We found that the 1 Hz stimulation and HFS protocolsequally antagonized CP-AMPARs (Fig. 3F). From these experi-ments, we conclude that CP-AMPARs do not regulate the STPand that HFS in the presence of IEM-1460 does not further an-

A B

C D E

Figure 1. Release probability and CP-AMPAR mediated synaptic transmission declines during MNTB principal cell maturation. A, Normalized EPSC peak amplitudes evoked at 1 Hz (downsampledto 0.05 Hz) plotted against time in representative recordings from P8 and P21 animals (open and closed black circles, respectively). EPSC amplitudes are shown in control conditions (time point 1),after the addition of IEM-1460 (60 �M; time point 2), and during washout (time point 3). External Ca 2� is 1.2 mM. The average normalized EPSC response recorded from P18 –P22 animals (n 6)in the presence of NBQX (1 �M, purple) is shown for comparison. � of the EPSC block was fit by a single exponential function for IEM-1460 (P8: �0.81 min; P21: �1.20 min) and NBQX (P18 –P22:� 0.43 min). B, Average from 20 EPSCs of the P8 (top) and P21 (bottom) animals at each designated time point in A displayed with the stimulus artifacts blanked for clarity. C, Representativenormalized EPSC amplitudes from P4, P9, P21, and P31 animals in control (black traces) and in the presence of IEM-1460 (red traces with peaks indicated by black arrows). D, Summary data of thepercentage block induced by application of IEM-1460 at the four developmental age ranges (one-way ANOVA with post hoc Tukey’s test; P4 –P5: n 4, P8 –P11: n 12, P18 –P22: n 11,P30 –P34: n 9; P4 –P5 vs P8 –P11: p 0.39, P4 –P5 vs P18 –P22: p � 0.0001, P4 –P5 vs P30 –P34: p � 0.0001, P8 –P11 vs P18 –P22: p � 0.0001, P8 –P11 vs P30 –P34: p � 0.0001, P18 –P22vs P30 –P34: p 0.44). E, Summary data of the � is shown for the four age groups (one-way ANOVA with post hoc Tukey’s test; P4 –P5 vs P8 –P11: p 0.97, P4 –P5 vs P18 –P22: p 0.93, P4 –P5vs P30 –P34: p0.042, P8 –P11 vs P18 –P22: p0.51, P8 –P11 vs P30 –P34: p0.009, P18 –P22 vs P30 –P34: p0.035). � reflects a weighted mean of low and high vesicle fusion probabilitiesat different active zones and was used as a measure of relative Pr. In the P4 –P5 age range, we observed uniformly high Pr values, whereas the adult-like P30 –P34 age range has a diversity of Pr valuesfrom high to low. ns, nonsignificant, *p � 0.05, ***p � 0.001.


tagonize the EPSCs compared with the 1 Hz stimulation proto-col, although HFS releases more glutamate overall.

Synaptically evoked Ca 2� transients in the principal cells ofthe MNTBOur EPSC data suggested that CP-AMPAR mediated transmis-sion declined upon synaptic maturation. We sought to confirmthis finding using Ca 2� imaging as an alternative approach. In aneffort to measure synaptically evoked CP-AMPAR-mediatedCa 2� transients in the MNTB principal cells, we performedwhole-cell recordings and included the Ca 2� indicator OGB1 inthe recording pipette. First, a short voltage-clamp step depolar-ization to �10 mV (�5 min after break-in) was applied to all cells

and used as a dye-loading control. This depolarizing pulse pro-duced a robust Ca 2� signal compared with quiescent (baseline)conditions (Fig. 4A). The principal cells were subsequently volt-age clamped at �70 mV. When applying midline afferent fiberstimulation at a frequency of 100 Hz for 500 ms, we detected a5.5 0.7% increase in fluorescence in the P8 –P11 group. Appli-cation of IEM-1460 to the slices blocked 54.2 5.3% of thisresponse, resulting in a 2.5 0.3% increase in fluorescence (Fig.4B1–B3). These data suggest that the Ca 2� signal detected wasprimarily due to Ca 2� influx through CP-AMPARs. When weperformed similar experiments in the P18 –P22 group, the syn-aptically evoked Ca 2� transients were greatly reduced (2.7

A

B D

E F

Figure 2. Blocking CP-AMPARs suppresses spontaneous mEPSC amplitudes. A, Representative mEPSCs recorded at a holding potential of �70 mV from a P8 (left) and a P20 (right) animal incontrol conditions (black) and after the addition of IEM-1460 (red). B, mEPSC peak amplitude is reduced in P8 –P11 and P18 –P22 animals after the addition of IEM-1460 (paired t test; P8 –P11: n 6, p � 0.0001; P18 –P22: n 6, p 0.0019). C, Frequency of the mEPSCs was not altered by IEM-1460 (paired t test; P8 –P11: n 6, p 0.1173; P18 –P22: n 6, p 0.1494). D, Percentageblock induced by IEM-1460 between the P8 –P11 and P18 –P22 groups was not different (unpaired t test; P8 –P11: n 6; P18 –P22: n 6; p 0.0955). E, F, Frequency distribution of mEPSC peakamplitudes (bin size 10 pA) for the P8 –P11 and P18 –P22 groups, respectively, in control conditions (black) or after the addition of IEM-1460 (red). The distribution of electrical noise is shownin gray. The total numbers of events per condition are shown in parentheses in the figure. ns, nonsignificant, **p � 0.005, ***p � 0.001.


0.6% increase in fluorescence). The addition of IEM-1460 hadno further effect on this response (IEM-1460: 2.8 0.6%increase in fluorescence; Fig. 4C1–C3). These data suggest thatCP-AMPAR-mediated synaptic transmission declines duringsynaptic maturation.

However, the results of these experiments left us with twomajor unresolved questions: (1) what process contributed to theremaining unblocked Ca 2� response in the P8 –P11 group? and(2) why was there no apparent contribution of CP-AMPARs tothe Ca 2� response in the P18 –P22 group, although we estimated�40% of the AMPAR population were CP-AMPARs by means ofelectrophysiology? We first tested whether the remaining compo-nent of the Ca 2� transient in the P8 –P11 group was mediated byacid-sensing ion channels (ASICs), which have recently beenshown to mediate a component of the synaptically evoked post-synaptic current and Ca 2� transient in the MNTB (Gonzalez-

Inchauspe et al., 2017). We tested the contribution of ASICs withthe pore blocker amiloride (Waldmann et al., 1997). We recordedEPSC trains while simultaneously imaging Ca 2� transients withOGB-1 in P8 –P11 animals (Fig. 5A1,A2). We found that IEM-1460 inhibited the first EPSC and that subsequent application ofIEM-1460 � amiloride (750 �M) further reduced the first EPSCamplitude by 28.5 3.3% without affecting STP (Fig. 5B–D).These data confirm that ASIC-mediated currents contribute to theEPSC (Gonzalez-Inchauspe et al., 2017). The Ca2� imaging simi-larly yielded Ca2� transients that could be inhibited by IEM-1460and, after the addition of IEM-1460 � amiloride, we found a further18.1 6.2% block of the Ca2� signal compared with IEM-1460alone (Fig. 5E1–E3). We thus conclude that Ca2� transients elicitedby afferent fiber stimulation in the immature MNTB principal cellsenter via two distinct routes: one route is through CP-AMPARs andthe other is through ASICs gated by proton release.

A

B

C D E F

Figure 3. CP-AMPARs do not regulate STP. A, EPSC trains evoked by midline stimulation at a frequency of 100 Hz were recorded in control conditions (black) and after application of IEM-1460(red). Representative traces are displayed for a P10 (left) and a P20 (right) animal. External Ca 2� is 1.2 mM. B, Ten sweeps (z-axis) of stimulation at 100 Hz (50 stimuli - x-axis) every 10 s yield EPSCswith peak amplitudes ( y-axis) displayed as surface plots for representative P10 and P20 recordings. The control condition (shown as sweep 1) was an average of �3 trains. The surfaces arepseudo-colored depicting red for greater amplitudes and purple for smaller. Note the greater block by IEM-1460 in each successive sweep compared with the control at P10, but not at P20. C, D,Summary of paired-pulse ratio (paired t test; P8 –P11: n 9, p 0.2554; P18 –P22: n 12, p 0.4875) and � (paired t test; P8 –P11: n 9, p 0.9521; P18 –P22: n 12, p 0.8049) beforeand after application of IEM-1460. E, Summary data of the first EPSC amplitude block by IEM-1460 (paired t test P8 –P11: n9, p0.0036; P18 –P22: n12, p0.0003). F, First EPSC percentageblock is stronger in the P8 –P11 group than in the P18 –P22 group using the 100 Hz blocking protocol (unpaired t test; P8 –P11: n 9; P18 –P22: n 12; p 0.0044). Additionally, the effect ofIEM-1460 on EPSC percentage block is similar using either the 1 Hz (from Fig. 1D) or 100 Hz stimulation paradigms in both age groups (unpaired t test P8 –P11: p 0.1339; P18 –P22: p 0.4171).ns, nonsignificant, **p � 0.005, ***p � 0.001.


Broadening mature presynaptic APs with TEATo address our second unresolved question as to why there wasan undetectable contribution of CP-AMPARs to the synapticallyevoked Ca 2� transients in the P18 –P22 group, we focused onthe presynaptic AP waveform. During postnatal development,the presynaptic AP width becomes briefer at the calyx ofHeld synapse, which underpins the progressively lower Pr

(Taschenberger and von Gersdorff, 2000). To control for thepossibility that presynaptic AP speeding was the underlyingcause of the apparent decrement in the number of CP-AMPARs after the onset of hearing, we pharmacologically ma-nipulated the mature presynaptic AP shape in P18 –P22animals to a waveform that more closely resembled that ofP8 –P11 animals. We used TEA to block potassium channels

and induce spike broadening and thus permit the entry ofmore Ca 2� ions to the presynaptic nerve terminal (Wang andKaczmarek, 1998; Johnston et al., 2010). We recorded HFSEPSC trains in control conditions or in the presence of TEA(100 �M; Fig. 6A). We observed an initial facilitation and milddepression of the control EPSC train for P18 –P22 in morephysiological 1.2 mM external Ca 2� (Hermann et al., 2007;Lorteije et al., 2009) and found that the first EPSC was poten-tiated and that the paired-pulse ratio was decreased by theapplication of TEA (Fig. 6 B, C). Using the Elmqvist and Quas-tel method to estimate readily releasable vesicle pool (RRP)size and Pr, a method reliant on the initial responses of astimulus train (Taschenberger et al., 2002), we found that TEAincreases presynaptic Pr, leaving the size of the RRP un-

A

B1 B2 B3

C1 C2 C3

Figure 4. CP-AMPAR-mediated Ca 2� transients in the MNTB principal cells are developmentally regulated. A, Representative MNTB principal cell loaded with the Ca 2� indicator OGB1 (50 �M)at �5 min after break-in at a holding potential of �70 mV during quiescent conditions (left) or during a short postsynaptic voltage depolarization to �10 mV (right). Scale bar, 10 �m. B1,CP-AMPAR mediated Ca 2� transients induced by afferent fiber stimulation at a frequency of 100 Hz for 500 ms (gray box denotes stimulation) in P8 –P11 animals during control conditions (black)or after application of IEM-1460 (red). The Ca 2� response observed during the voltage-step (green) is included for comparison. The region outlined by dotted lines is shown at higher magnificationin B2. B3, Summary data of the peak Ca 2� responses in the P8 –P11 group (paired t test; n 9, p 0.0008). C1, Ca 2� transients recorded as in B1 but in P18 –P22 animals. The region outlinedby dotted lines is shown at higher magnification in C2. C3, Summary data of the peak Ca 2� responses in the P18 –P22 group (paired t test; n 7; p 0.79). ns, nonsignificant, ***p � 0.001.


changed (Elmqvist and Quastel, 1965; Neher, 2015; Fig. 6D1–D3). We observed a high heterogeneity of Pr (range 0.05– 0.25)and RRP size at these P18 –P22 calyx synapses, which mayreflect distinct calyceal morphologies and the diverse Ca 2�

influx at stalks and swellings (Grande and Wang, 2011; Spirouet al., 2008; cf.Figs. 1E, 6D3).

We next investigated whether IEM-1460 application simi-larly antagonized the CP-AMPARs of P18 –P22 synapses in thecontinued presence of TEA as in control conditions. We re-corded HFS EPSC trains and blocked CP-AMPARs with IEM-

1460 (Fig. 7A). We found that the addition of IEM-1460 toslices in the continued presence of TEA reduced the amplitudeof the first EPSC (Fig. 7B) and produced an equal percentageblock of the first EPSC compared with those experiments per-formed in the presence of IEM-1460 alone (Fig. 7C). Thesedata confirm that the apparent reduction in CP-AMPARspresent in mature P18 –P22 animals is not an artifact of pre-synaptic AP speeding. Additionally, these data confirm thatour IEM-1460-blocking protocol in the absence of TEA satu-rated CP-AMPAR block because increasing presynaptic gluta-

B C D

E1 E2 E3

Figure 5. ASIC-mediated synaptic currents and Ca 2� transients in the juvenile MNTB Principal cell. A1, Representative EPSC traces (100 Hz for 500 ms, left) evoked by midline stimulation aredisplayed from a P8 animal in control conditions (black), after the addition of IEM-1460 (red, 60 �M), and after the addition of IEM-1460 (60 �M) � amiloride (blue, 750 �M). The first EPSC fromthe train is enlarged in A2 for comparison and the summary data for their amplitude is shown in B (one-way ANOVA with post hoc Tukey’s test; n 7; control vs IEM-1460: p 0.0046, control vsIEM-1460 � amiloride: p 0.0034, IEM-1460 vs IEM-1460 � amiloride: p 0.0081). C, D, Neither IEM-1460 nor IEM-1460 � amiloride application altered the STP parameters of paired-pulseratio (C: n 7, one-way ANOVA with post hoc Tukey’s test, control vs IEM-1460: p 0.4352, control vs IEM-1460 � amiloride: p 0.7354, IEM-1460 vs IEM-1460 � amiloride: p 0.7217) or� (D: control vs IEM-1460: p 0.2928, control vs IEM-1460 � amiloride: 0.7719, IEM-1460 vs IEM-1460 � amiloride: p 0.4437). E1, Ca 2� transients in the postsynaptic MNTB principal cellswere measured using OGB1 evoked by afferent fiber stimulation at a frequency of 100 Hz for 500 ms (gray box denotes stimulation) in control conditions (black), after the addition of IEM-1460 (red),and after the addition of IEM-1460 � amiloride (blue). The green trace represents the Ca 2� transient from a voltage step. The region outlined by dotted lines in E1 is shown at higher magnificationin E2. Application of IEM-1460 � amiloride further reduced the MNTB principal cell Ca 2� transient compared with IEM-1460 alone. E3, Summary data of the peak Ca 2� responses in the P8 –P11group (one-way ANOVA with post hoc Tukey’s test, n 7; control vs IEM-1460: p 0.0068, control vs IEM-1460 � amiloride: p 0.0068, IEM-1460 vs IEM-1460 � amiloride: p 0.0445). ns,nonsignificant, *p � 0.05, **p � 0.005.


mate release with TEA did not increase the CP-AMPARpercentage block.

We next investigated whether TEA-induced spike broaden-ing, which potentiates presynaptic glutamate release and acti-vates more postsynaptic AMPARs, would lead to detectable Ca 2�

transients in the principal cells of the MNTB. We includedOGB-1 in the recording pipette and performed whole-cell re-cordings on principal cells in the P18 –P22 group in an effort todetect CP-AMPAR-mediated Ca 2� transients in the continuedpresence of TEA. Even with TEA, afferent fiber stimulation onlyproduced a 2.6 1.3% increase in fluorescence in the postsyn-aptic compartment and application of IEM-1460 did notchange this response (IEM-1460: 2.7 1.1% increase in fluo-rescence; Fig. 7D1–D3). To further investigate this, we usedP27–P30 animals, excluded EGTA completely from the OGB-1-loading pipette, and increased the 100 Hz afferent fiberstimulation duration to 1 s. Under these conditions, we de-tected a 4.5 0.7% increase in fluorescence in the principalcells of the MNTB in response to synaptic stimulation. Appli-cation of IEM-1460 to the slices blocked 33.4 17.3% of thisresponse, resulting in a 2.6 0.4% increase in fluorescence(Fig. 8A1–A3). These data confirm that functional CP-AMPARs are present at the more mature P27–P30 principalcells of the MNTB, but in reduced numbers.

CP-AMPAR regulation of postsynaptic spiking fidelityWe next sought to test the functional contribution of CP-AMPARs to postsynaptic spiking. We recorded HFS AP trains incurrent-clamp mode for control conditions or after the additionof IEM-1460 (Fig. 9A). These experiments were performed at PT(33–35°C) to elicit HFS (100 Hz) without failures. They were alsodone in the continued presence of DL-AP5 (50 �M) to inhibitNMDA receptors (Taschenberger and von Gersdorff, 2000; Futaiet al., 2001). Moreover, the principal cells were current injected toa resting membrane potential of ��70 mV throughout the en-tirety of the recording (Fig. 9B). We found that inhibiting CP-AMPARs produced a significant increase in the frequency of APfailures in the P8 –P11 group (control: 0.2 0.2%; IEM-1460:35.5 13.6%), whereas no effect was observed in the P18 –P22group (control: 2.3 1.4%, IEM-1460: 4.1 2.2%; Fig. 9C). Weadditionally analyzed several parameters of the first AP of thetrain (Fig. 9D). We found that IEM-1460 inhibited the peak am-plitude of the postsynaptic MNTB principal cell AP at both ageranges, albeit more strongly in the P8 –P11 group (Fig. 9E). Inaddition, IEM-1460 reduced the depolarizing afterpotential sig-nificantly for the P8 –P11 group (Fig. 9D), which is probably dueto the slow decay of EPSCs (Fig. 1C; Johnston et al., 2009) and theslow discharge of the unmyelinated axon capacitance (Borst et al.,1995; Kim et al., 2010). The AP half-width remained unaffectedin both age groups, whereas the AP peak delay was increased in

A B C

D1 D2 D3

Figure 6. TEA-induced presynaptic spike broadening potentiates synaptic vesicle release. A, EPSCs were evoked by midline stimulation in a P21 animal at a frequency of 100 Hz for 500 ms andthe peak amplitude is plotted as a function of time in control conditions (black) or in the presence of TEA (red, 100 �M). Cell-matched recordings were not performed for every data point. B, Summarydata for the first EPSC amplitude is displayed and the EPSCs were potentiated in the presence of TEA (unpaired t test; control: n 12; TEA: n 8, p 0.0134). C, Paired-pulse ratio was reduced(unpaired t test; control: n 12; TEA: n 8, p 0.0186). D1, The Elmqvist and Quastel method was used to estimate presynaptic Pr and RRP size. The individual release (EPSC amplitudes duringthe stimulus train) was plotted as a function of the cumulative release (sum of EPSCs) in control (black) or in TEA (red). D2, Size of the RRP was unchanged in TEA (unpaired t test; control: n 12;TEA: n 8, p 0.7858). D3, Presynaptic Pr was increased in the presence of TEA (unpaired t test; control: n 12; TEA: n 8, p 0.0406). Note that control synapses display a high heterogeneityof RRP size and a diverse range of Pr values from 0.05 to 0.25 for P18 –P22 calyx of Held nerve terminals. ns, nonsignificant, *p � 0.05.


both ages (Fig. 9F,G). These data support the notion that CP-AMPARs play a major role in triggering the postsynaptic APbefore the onset of hearing given that application of IEM-1460had a profound effect on the rate of AP failures. However, theCP-AMPARs appear to play a very minor role in spike timing andno role in failures during 100 Hz stimulation at more matureP18 –P22 calyx of Held synapses.

DiscussionWe have shown that IEM-1460 blocks �80% of the EPSC ampli-tude in immature P4 –P5 calyx synapses. CP-AMPARs thus play amajor role during the period right after the single, large calyxsynapse is established and strengthened on the principal cell soma(Hoffpauir et al., 2009). The expression of CP-AMPARs is thenprogressively downregulated. Our data show that 1 Hz stimula-

A B C

D1 D2 D3

Figure 7. CP-AMPAR-mediated Ca 2� transients are undetectable in mature principal cells recorded in the presence of TEA. A, Midline stimulation was used to evoke EPSCs at 100 Hz for 500 msin the presence of TEA (black) or in TEA � IEM-1460 (red). B, Addition of IEM-1460 reduced the first EPSC peak amplitude (paired t test: n 8, p 0.0004). C, In the P18 –P22 group, the percentageblock induced by the addition of IEM-1460 alone (Figs. 1D, 3F ) was compared with that recorded in the presence IEM-1460 � TEA. No difference was detected (unpaired t test; IEM-1460: n 23;IEM-1460 � TEA: n 8, p 0.1141). D1, OGB1 was used to simultaneously record Ca 2� transients from the principal cells of the MNTB induced by midline stimulation (gray box denotesstimulation) at a holding potential of�70 mV. The average responses are presented in the presence of TEA (black) and TEA� IEM (red). The Ca 2� response induced by a postsynaptic depolarizationin TEA (green) is shown for comparison. D2, Region outlined by dotted lines in D1 at higher magnification. Ca 2� transients evoked by midline stimulation were unchanged in the presence of TEAand TEA � IEM. D3, Summary data of the peak Ca 2� responses in the P18 –P22 group recorded in the presence of TEA and TEA � IEM (paired t test; n 5, p 0.6370). ns, nonsignificant, ***p �0.001.

A1 A2 A3

Figure 8. Increasing synaptic drive and limiting EGTA in the loading pipette unveils CP-AMPAR-mediated Ca 2� transients in mature principal cells. A1, OGB1 was used to record Ca 2� transientsfrom the principal cells of the MNTB induced by midline stimulation at a frequency of 100 Hz from P27–P30 mice. EGTA was excluded from the loading pipette and the stimulation time was increasedto 1 s. Midline stimulation (gray box denotes stimulation) produced a Ca 2� response (control, black) and application of IEM-1460 inhibited this response (IEM, red). The Ca 2� response to a 500 mspostsynaptic voltage-clamp step depolarization is shown for comparison (green). A2, Region outlined by dotted lines in A1 shown at higher magnification. A3, Summary data of the peak Ca 2�

responses recorded in the P27–P30 group in control conditions or after the addition of IEM-1460 (paired t test; n 8, p 0.0170). ns, nonsignificant, *p � 0.05.


tion produces a progressive, activity-dependent block of theEPSCs by IEM-1460, consistent with recent cryo-EM mechanis-tic studies of open channel block of CP-AMPARs by adamantanederivatives and spider toxins (Twomey et al., 2018). Importantly,the EPSC amplitude measurements were taken after only 40 s ofcontinuous 1 Hz stimulation. The contributions of AMPAR de-

sensitization to short-term synaptic de-pression are minimal using this low-frequency 1 Hz stimulation (Wong et al.,2003; Renden et al., 2005). Additionally,we were able to obtain a complete recov-ery from the effect of IEM-1460, whichsuggests that our results are not influ-enced by synaptic rundown. The reduc-tion in the EPSC at P18 –P22 (40.8 2.7%) is close to that reported previouslyin P14 mice (43%) using a different CP-AMPAR antagonist (Joshi et al., 2004).We confirmed that the blocking efficiencyof IEM-1460 was maximal with continu-ous 1 Hz stimulation because increasingthe stimulation frequency to 100 Hz didnot further inhibit the amplitude of theEPSCs.

Spontaneous mEPSCs andAP-evoked EPSCsWe found that mEPSCs and EPSCsevoked by afferent fiber stimulation werecomparably inhibited by IEM-1460. Be-cause the EPSCs were reduced by 68% and41% at P8 –P11 and P18 –P22, respec-tively, we predicted that the average am-plitude of the mEPSCs in IEM-1460would be �22 pA at P8 –P11 and 44 pA atP18 –P22 based on our control mEPSCmeasurements. Accordingly, when weperformed Gaussian function fits on ourpeak amplitude distributions and con-strained the mean of the fits to the afore-mentioned predicted values, we obtainedcurves that closely approximated the peakamplitude distributions. Because ourcurve fit predictions closely matched ourmEPSC amplitude distributions, thesedata suggest that the AMPARs mediatingmEPSCs and evoked EPSCs are indistin-guishable from one another with respectto AMPAR subunit composition (Kava-lali, 2015; Tsintsadze et al., 2017).

Postsynaptic Ca 2� imagingduring developmentTo record Ca 2� transients, the MNTBprincipal cells were continuously held at�70 mV, thereby limiting the contribu-tions of NMDA and voltage-gated Ca 2�

channels to the Ca 2� influx (Bollmann etal., 1998). Additionally, maintaining thecell at a holding potential of �70 mV pro-duced a constant and high driving forcefor Ca 2� ion passage through open CP-AMPARs. We excluded polyamines from

the recording pipette in an effort to limit their possible antago-nistic effects on CP-AMPARs (Bowie and Mayer, 1995). We weresurprised to observe significant CP-AMPAR-mediated responsesin the P8 –P11 group because AMPAR desensitization to 100 Hzstimulation is quite robust at this age (Renden et al., 2005). De-sensitization may indeed limit Ca 2� flux by CP-AMPARs. How-

A

B C

D

E F G

Figure 9. Inhibiting CP-AMPARs impairs synaptic fidelity in juvenile principal cells, but not in mature principal cells. A, Repre-sentative AP trains elicited by 500 ms midline stimulation at 100 Hz recorded from a P9 animal (top) and a P22 animal (bottom) incontrol conditions (black) or after the addition of IEM-1460 (red). All recordings were performed at a PT of 33–35°C and DL-AP5 (50�M) was included in the extracellular solution to inhibit NMDA receptors. B, Cells were current injected to normalize restingmembrane potential to ��70 mV (paired t test; P8 –P11: n 7, p 0.7152; P18 –P22: n 7, p 0.3451). C, IEM-1460increases AP failures in the P8 –P11 group, but not in the P18 –P22 group (paired t test; P8 –P11: n 7, p 0.0396; P18 –P22:n 7, p 0.1494). D–G, First AP from the representative traces shown in A used to compare several parameters (D; tested usingpaired t test; P8 –P11: n7; P18 –P22: n7) of the AP waveform such as AP peak amplitude (E; P8 –P11: p0.0062; P18 –P22:p 0.0214), half-width (F; P8 –P11: p 0.1246; P18 –P22: p 0.6696), and AP delay (G; P8 –P11: p 0.0184; P18 –P22: p 0.0136). ns, nonsignificant, *p � 0.05, **p � 0.005.


ever, we opted not to use cyclothiazide (CTZ) to blockdesensitization because of its effects on presynaptic Pr (Ishikawaand Takahashi, 2001), although the inclusion of CTZ does pro-duce stronger cobalt uptake via CP-AMPARs (Eybalin et al.,2004).

Using confocal laser imaging, we were unable to detect aneffect of IEM-1460 on the CP-AMPAR-mediated Ca 2� transientsin the P18 –P22 group in both the absence and presence of TEA,although we predicted from our EPSC recordings that likely 30 –40% of the AMPARs are permeable to Ca 2�. However, uponremoval of 0.5 mM EGTA from the patch pipette and increasingthe stimulation time to 1 s at a frequency of 100 Hz, we were ableto observe a clear effect of IEM-1460 on the Ca 2� transients.These Ca 2� transients may be difficult to observe in the matureMNTB principal cells because the postsynaptic AMPARs prefer-entially cluster in progressively smaller areas during development(Hermida et al., 2010). Therefore, the Ca 2� flux may only exist insmall microdomains under the synaptic cleft. Additionally, Ca 2�

buffering becomes stronger and faster in the principal cell withage because several different types of Ca 2� binding proteins areupregulated with development (Lohmann and Friauf, 1996;Felmy and Schneggenburger, 2004).

Synaptic AMPAR subunits change during developmentThe temporal expression patterns of GluA2 in the MNTB, thecritical regulator of Ca 2� permeability, are not in completeagreement in the literature. Developmental studies of MNTBsuggested that GluA2 levels are quite high throughout the firsttwo postnatal weeks and decline rapidly during the third postna-tal week (Caicedo and Eybalin, 1999). However, a subsequentstudy suggests GluA2 is low during early postnatal developmentand is likely not necessary for MNTB synaptogenesis (Joshi et al.,2004). Our results in P4 –P5 synapses agree with this particularfinding. Conversely, measurements of the mRNA levels codingfor the GluA2 subunit suggest that GluA2 is actually the secondmost abundant subunit and that these mRNA levels remain stablethroughout development (Koike-Tani et al., 2005).

The GluA4 subunit is indispensable for the high-fidelity trans-mission of the calyx of Held synapse (Joshi et al., 2004; Yang et al.,2011). However, our results are in contrast to those of Joshi et al.(2004) suggesting high Ca 2� permeability through the AMPARsafter hearing onset. Their conclusion is supported by rectifyingI–V relationships at P13–P14 and by the lack of immunohisto-chemical staining for GluA2 upon maturation. Although the rec-tification index is generally agreed upon to be a measure ofAMPAR subunit composition, the sensitivity as a readout forCa 2� permeability remains controversial. In hippocampal in-terneurons, the amount of GluA2 necessary to abolish IV rectifi-cation in AMPARs is higher than that needed to abolish Ca 2�

permeability (Washburn et al., 1997). Accordingly, 50% of theinterneurons that possessed rectifying I–Vs were also positive forGluA2 (Washburn et al., 1997). Conversely, in retinal bipolarcells of salamander and auditory cochlear nucleus chick neurons,the EPSC I–V curves were linear while retaining high divalent ionpermeability (Gilbertson et al., 1991; Otis et al., 1995), althoughin these experiments, the investigators did not add polyamines tothe patch pipette. Therefore, using the rectification index ob-tained from I–V plots of EPSCs may not be an accurate readoutfor the Ca 2� permeability of AMPARs in all scenarios because thestructural components that mediate Ca 2� permeability and poly-amine block are different.

Diversity of AMPARs and release probability at auditorybrainstem nucleiCP-AMPARs are found in a subclass interneurons in the neocor-tex, where they regulate the dynamic balance of excitation andinhibition (Hull et al., 2009; Lalanne et al., 2016). At the innerhair cell synapse of the cochlea, a ribbon-type synapse, CP-AMPARs are expressed at the afferent fibers before the onset ofhearing and their expression declines with maturation (Eybalin etal., 2004; Reijntjes and Pyott, 2016), although some receptorsmay remain in mature fibers (Sebe et al., 2017). Likewise, in thesuperior paraolivary nucleus of mice, a mixed population of CP-AMPARs and Ca 2�-impermeable AMPARs were present beforehearing onset; however, upon maturation, the population wascomposed primarily of Ca 2�-impermeable AMPARs (Felix andMagnusson, 2016). However, in the same study, it was concludedthat neurons of the lateral superior olive retain a mixed popula-tion of CP-AMPARs and Ca 2�-impermeable AMPARs through-out development (Felix and Magnusson, 2016). Indeed, manyregions in the auditory brainstem have high expression of CP-AMPARs (Otis et al., 1995; Ravindranathan et al., 2000; Gardneret al., 2001; Takago and Oshima-Takago, 2018).

Several factors can modulate the Ca 2� permeability of theAMPAR past the level of GluA2 subunit inclusion. For example,the functional consequence of auxiliary proteins on the biophys-ical properties of AMPARs have been discovered only recently(Bowie, 2018). In addition to the regulation of AMPAR traffick-ing, these proteins can bidirectionally modulate single-channelunitary conductance, Ca 2� permeability, and EPSC rectification(Wollmuth, 2018). The auxiliary subunits stargazin and cor-nichon can increase Ca 2� permeability (Kott et al., 2009;Coombs et al., 2012), whereas the recently discovered auxiliarysubunit GSG1L reduces Ca 2� permeability (McGee et al., 2015).The developmental expression profile of the AMPAR auxiliarysubunits in the auditory brainstem remains unknown and awaitsfuture studies.

Our results suggest that P4 –P5 synapses have a high Pr andlow abundance of synaptic GluA2, making them highly Ca 2�

permeable, whereas adult-like P30 –P34 synapses are more di-verse and have on average lower Pr. We propose that maturesynapses express both GluA2 and GluA4 subunits, making theirAMPARs fast and impermeable to Ca 2�. Changes in spontane-ous and patterned spike activity during postnatal developmentmay propel higher expression levels of GluA2 and lower Pr values(Tokuoka and Goda, 2008; Babola et al., 2018), whereas sound-driven spiking activity may subsequently stabilize GluA2 expres-sion and short-term plasticity (Hermann et al., 2007; Ngodup etal., 2015). Loss of CP-AMPARs seems to coincide with the down-regulation of NMDARs over the critical developmental windowof hearing onset (Taschenberger and von Gersdorff, 2000; Futaiet al., 2001). It appears that limiting postsynaptic Ca 2� flux isimportant for high-fidelity transmission at this synapse. A sparseexpression of CP-AMPARs and NMDARs at mature synapsesmay also reduce the likelihood of excitotoxicity associated withCa 2� influx. An AMPAR subunit combination with a predomi-nant ratio of GluA2:GluA4 near 1:3 thus represents an attractivepossible scenario: Ca 2� permeability is blocked, whereas fast gat-ing kinetics, high unitary conductance, and faster recovery fromdesensitization are maintained.

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